PROJECT SUMMARY Chronic hepatitis B virus (HBV) infection is associated with an ineffective T cell response that fails to control the virus. While the precise mechanism of T cell dysfunction in chronic hepatitis B is not well understood, prior studies using blood and liver tissue from acute and chronic HBV patients have identified a number of critical immunological mechanisms that may contribute to this poor immune response. These mechanisms include the killing of HBV-specific T cells by liver-resident natural killer (NK) cells, a general tolerogenic environment in the liver that is manifested by immunosuppressive immune cells, cytokines, and metabolic pathways, and the loss of T cell function through inhibitory receptor signaling pathways and exhaustion. The study of HBV-host interactions and the development of new curative immunotherapies for chronic hepatitis B have been hampered by the lack of physiological animal models for chronic infection. A new mouse model of HBV replication was recently described that is based on adeno-associated virus (AAV)-mediated transduction of the liver with the HBV genome, which leads to persistent virus replication and immune tolerance to HBV antigens. Compared to other mouse models of HBV replication, the AAV-HBV model has important advantages, including physiological intrahepatic immune priming, technical ease and reproducibility, peripheral T and B cell tolerance to HBV, and the ability to study virus elimination from the liver. Using this model, we will test the hypothesis that HBV therapeutic vaccine efficacy can be enhanced by manipulating mechanisms of immune dysfunction to promote viral clearance. Our general approach will be to combine AAV-HBV transduction with genetically modified mice and antibody-mediated depletion/blockade to evaluate the importance of immune cells, cytokines, and regulatory pathways for eliciting functional CD8+ T cell responses and controlling HBV following therapeutic immunization. To evaluate our hypothesis, we will carry out three specific aims. First, we will assess the inhibition of HBV- specific T cells by NK cells. Second, we will define the role of regulatory cells, cytokines, and metabolic pathways. Third, we will evaluate T cell inhibitory receptor function and exhaustion. The knowledge that will be gained from this research will impact the field in two important ways. First, it will provide new mechanistic insight into the relationship between T cell dysfunction and HBV persistence, and form a strong foundation for future studies using the AAV-HBV model to better understand the virus-host interactions that are observed in humans. Second, it will reveal new immunotherapeutic targets for potential approaches to cure chronic HBV infection.